Imipenem hydrolysis by metallo-beta-lactamase BcII: hints into the catalytic mechanism

MARIANA TIONI, LETICIA LLARRULL, BRIAN BENNETT, ALEJANDRO J. VILA

Montevideo-Uruguay

Congreso; 6th International Conference of Biological Physics ICBP 2007, 5th Southern Cone Biophysics Congress and 34th Annual Meeting of the Argentinean Biophysical Society; 2007

Sociedad Argentina de Biofisica

β-lactamases constitute the major bacterial defense mechanism against β-lactam antibiotics.These enzymes hydrolyse the β-lactam ring rendering the antibiotics ineffective. Metallo- β-lactamases (MβLs) comprise a group of β-lactamases that require Zn(II) for function, in contrastto the well-known Serine-β-lactamases (SβLs) whose activity relies on a serine residue in theactive site. MβLs are considered a great threat for human health since they have a widesubstrate spectrum and cannot be inhibited by therapeutically useful drugs, effective againstSβLs. Rational design of such drugs relies on the thorough knowledge of the chemical nature ofthe hydolysis intermediates.Imipenem belongs to the family of carbapenems, the last generation of β-lactam antibiotics. Incontrast to SβLs, MβLs are efficient carbapenemases. Generally, imipenem hydrolysis givesrise to a mixture of species with a cleaved β-lactam ring and a rearranged double bond. Wehave studied the reaction of BcII, the Bacillus cereus MβL, with imipenem. We found that BcII isstereospecific rendering only one major hydrolysis product.We used stopped-flow and rapid freeze quench EPR (RFQ-EPR) experiments to study in detailthe catalytic behaviour of BcII. We used Co(II) as a spectroscopic probe for Zn(II) in order tocharacterise reaction intermediates and delineate a minimal kinetic pathway for imipenemhydrolysis by BcII. With both techniques, we could detect transient species with distinctivespectral features.Rapid kinetics sudies revealed that BcII uses a branched mechanism for imipenem hydrolysisand that a stable enzyme-product adduct is formed. Direct evidences of the formation of suchan adduct were obtained with UVVis, EPR and NMR spectroscopy.